Abstract Despite the vast excess of cellular RNAs, a single viral genomic RNA (gRNA) dimer is selectively packaged into new human immunodeficiency virus type 1 (HIV-1) particles. This occurs, in part, due to specific interactions between the HIV-1 Gag precursor protein and the gRNA psi (?) packaging signal. However, the precise mechanism of selective gRNA packaging in any retroviral system is still unknown. The multi-domain Gag precursor protein includes two nucleic acid-binding domains: matrix (MA) and nucleocapsid (NC). Binding of the NC domain to psi is necessary for efficient gRNA packaging, but the mechanism by which NC selectively interacts with psi is unclear. Moreover, based on recent in vitro and cell-based results, whether the specificity of psi RNA vs non-psi RNA binding by NC is sufficient for selective packaging has been called into question. In addition, the role of the MA domain in cellular and viral RNA binding and regulation of specific gRNA packaging and membrane targeting has been an active area of recent investigation. However, many open questions remain regarding the precise role of MA in selective gRNA packaging. The proposed work will address many of these important open questions and lead to an improved understanding of how selective gRNA packaging is achieved in retroviruses, providing new targets for anti-retroviral therapy. The overarching hypothesis of this proposal is that Gag-psi RNA interaction induces a Gag conformation that is optimal for Gag-Gag, Gag-gRNA, and Gag-membrane interaction, which collectively lead to selective gRNA packaging. The specific aims are: (1) To probe the specificity of retroviral Gag binding to psi-containing and non-psi RNAs, and (2) To probe the conformational dynamics, structure, and stoichiometry of HIV-1 Gag-RNA complexes.